The present invention relates to haptens and conjugate labels having improved specificity for anti-cortisol antibodies, methods for their preparation and use in immunoassays for the detection of cortisol. More particularly, the present invention relates to conjugates comprising horseradish peroxidase and reduced cortisol.
Cortisol is the major glucocorticoid in humans. It is synthesized and secreted by the zona fasciculata and the zona reticularis of the adrenal cortex. It is involved in the regulation of carbohydrate, protein, and lipid metabolism. Cortisol levels can rise ten fold following surgery or other major trauma, as the steroid acts to prevent vascular collapse, reduce inflammation, and suppress the immune system.
There are three primary medical disorders associated with hyperadrenalism: Cushing""s syndrome, hyperaldosteronism, and congenital adrenal hyperplasia. Cushing""s syndrome is the term used to describe any condition resulting from an increased concentration of circulating glucocorticoid, usually cortisol (Clinical Chemistry: Theory, Analysis, and Correlation; Lawrence A. Kaplan, Amadeo J. Pesce, C V Mosby Company, 1989, pp 673-4).
The detection and quantification of cortisol in human serum, plasma or urine is required for proper diagnosis, treatment and follow-up of cortisol related conditions.
Competitive binding immunoassays for cortisol comprise anti-cortisol antibody, usually bound to an immobilized or immobilizable substrate and labeled cortisol, or labeled analogs (derivatives) of cortisol. It shall be understood that whenever reference is made to labeled cortisol, unless otherwise indicated, the term is intended to encompass labeled analogs (derivatives) of cortisol. Labeled cortisol competes with cortisol for a limited number of anti-cortisol antibody binding sites. Signal derived from free or bound labeled cortisol is determined as a measure of the amount of cortisol.
The sensitivity and specificity of an immunoassay for cortisol are dependent on the labeled cortisol. It is important that labeled cortisol effectively compete for the limited number of anti-cortisol binding sites with steroids structurally similar to cortisol that may be present in a sample. Otherwise, a clinically acceptable determination of the amount of cortisol in the sample will not be obtained.
Individuals having a deficiency of the enzyme 11xcex2-hydroxylase, or who receive metyrapone will have greatly increased levels of 11-deoxycortisol, which is structurally similar to cortisol (Fundamentals of Clinical Chemistry, Tietz,N. W., W. B. Saunders Co., 1987,p 569) and potentially can compete with labeled cortisol for binding to anti-cortisol antibody. Other cortisol-like steroids that may be present in a sample, which potentially can compete with labeled cortisol for anti-cortisol antibody, include prednisolone, cortisone, and corticosterone. Such competition with labeled cortisol for anti-cortisol antibody is termed cross-reactivity.
Commercial cortisol assays can exhibit cross-reactivity with all the above-identified steroids. For example, a cross-reactivity with 11-deoxycortisol of greater than 10 percent has been observed; seriously compromising the accuracy of the assay for cortisol.
The problems associated with prior art assays for cortisol have been overcome using the conjugate labels described hereinbelow.
The present invention relates to compositions comprising novel reduced cortisol conjugates, methods for their preparation and use in immunoassays for cortisol.
In another aspect, it relates to conjugates of reduced cortisol as immunogens or haptens for eliciting anti-cortisol or anti-reduced cortisol antibodies.
It was found unexpectedly that labeled reduced cortisol conjugates of the present invention effectively compete with cortisol-like steroids for binding to anti-cortisol antibodies, thereby exhibiting significantly less cross-reactivity compared with prior art labeled cortisol conjugates. Immunoassays for cortisol comprising labeled reduced cortisol conjugates of the present invention exhibit both improved specificity and sensitivity for the determination of cortisol.
Accordingly, the present invention provides a reduced cortisol conjugate of formula: 
wherein X is O, S, sufonyl, or phosphono; X1 is a labeled or unlabeled natural or synthetic polymer or a label; Y1 is a linking group or a bond; X2 is a labeled or unlabeled natural or synthetic polymer or a label; Y2 is a linking group or a bond; A1 and A2 are each hydrogen or A1 and A2 together form a single bond, B1 and B2 are each hydrogen or B1 and B2 together form a single bond, E1 and E2 are each hydrogen or E1 and E2 together form a single bond provided at least one of A1 and A2, or B1 and B2, or E1 and E2 are each hydrogen.
In another aspect, the present invention relates to methods for the preparation of reduced cortisol conjugates. Accordingly, we provide a method for preparing a reduced cortisol conjugate of formula 
wherein X, X1, Y1, X2, Y2, A1, A2, B1, B2, E1 and E2 are as defined above, comprising:
reacting a compound of formula 
xe2x80x83with a reducing agent.
An alternative method is provided for preparing a reduced cortisol conjugate of formula 
wherein X, X1, Y1, X2, Y2, A1, A2, B1, B2, E1 and E2 are as defined above, comprising the steps of:
(i) reacting a compound of formula 
xe2x80x83with a reducing agent, thereby forming compound IA or IC; and
ii) with a first coupling agent, thereby forming a compound of formula 
xe2x80x83wherein G1 is a coupling group;
(iii) optionally, reacting X2 with a second coupling agent, thereby forming X2xe2x80x94G2, wherein G2 is a coupling group capable of forming a covalent bond with the coupling group G1, and wherein G1 and G2 may be the same;
(iv) optionally reacting compound IA or IC with X2xe2x80x94G2 wherein G2 is capable of forming a covalent bond with a functional group of X1, thereby forming reduced cortisol conjugate IB or ID;
(v) optionally reacting compound IIIA or IIIC with X2 wherein G1 is capable of forming a covalent bond with a functional group of X2, thereby forming reduced cortisol conjugate IB or ID;
(vi) optionally reacting compound IIIA or IIIC with X2xe2x80x94G2, thereby forming reduced cortisol conjugate IB or ID.
In yet another aspect, the present invention relates to methods for the qualitative or quantitative determination of cortisol that utilize the novel labeled reduced cortisol conjugates.
Accordingly, we provide a method for performing a competitive assay for cortisol comprising the steps of:
A) contacting a sample suspected of containing cortisol with
(i) an immobilized or immobilizable receptor that binds cortisol, thereby forming cortisol that is bound and cortisol that is not bound to the immobilized or immobilizable receptor,
(ii) a labeled reduced cortisol conjugate of formula IA, IB, IC or ID as defined above, thereby forming labeled reduced cortisol conjugate that is bound and labeled reduced cortisol conjugate that is not bound to the immobilized or immobilizable receptor;
B) detecting either the labeled reduced cortisol conjugate that is bound or the labeled reduced cortisol conjugate that is not bound to the immobilized or immobilizable receptor as a measure of the amount of cortisol in the sample.
In an alternative ebodiment, the assay method described above may be combined with a step wherein the labeled reduced cortisol conjugate that is bound is separated from the labeled reduced cortisol conjugate that is not bound to the immobilized or immobilizable receptor;
An alternative method for performing a competitive assay for cortisol using a dry analytical element is also provided, the dry analytical element comprising
a) a spreading zone,
b) one or more reagent zones,
c) a support, and
together or separately in one or more of the zones, an immobilized receptor that binds cortisol and optionally, a labeled reduced cortisol conjugate of formula IA, IB, IC or ID defined above, wherein the method comprises the steps of:
A) contacting the spreading zone of the dry analytical element with
i) a sample suspected of containing cortisol, thereby forming cortisol that is bound and cortisol that is not bound to the immobilized receptor,
ii) labeled reduced cortisol conjugate if it is not present in the dry analytical element, thereby forming labeled reduced cortisol conjugate that is bound and labeled reduced cortisol conjugate that is not bound to the immobilized receptor,
B) optionally, separating the labeled reduced cortisol conjugate that is bound from the labeled reduced cortisol conjugate that is not bound to the immobilized receptor; and
C) detecting either the labeled reduced cortisol conjugate that is bound or the labeled reduced cortisol conjugate that is not bound to the immobilized receptor as a measure of the amount of cortisol in the sample.
In another sense, the present invention relates to haptens or immunogens of formula IA, IB, IC or ID as defined above and compositions comprising the immunogens. It also relates to methods of producing anti-cortisol antibodies using the immunogens of the present invention, by immunizing a host animal, removing blood from the host, and separating antibodies that bind cortisol from the host animal""s blood serum or plasma. In another related method, the spleen, thymus or other organ that is populated with antibody producing cells is removed from the immunized host animal, antibody secreting hybridomas are prepared using antibody producing cells of the spleen, thymus or other organ so removed, and antibodies that bind cortisol are selected therefrom.
In yet another sense, the present invention relates to methods of reducing cross-reactivity in immunoassays for cortisol using labeled reduced cortisol conjugates of formula IA, IB, IC or ID.
The labeled reduced cortisol conjugates of the present invention, as stated, effectively compete with cortisol-like steroids for binding to anti-cortisol antibodies, thereby exhibiting significantly less cross-reactivity compared with prior art labeled cortisol conjugates.
The invention is described in detail with respect to a specific reduced cortisol conjugate comprising bovine serum albumin and horseradish peroxidase. This has been done to illustrate the present invention and is not intended in any way to limit the invention to this specific example. Other reduced cortisol conjugates, their syntheses and use as immunogens, or as reduced cortisol labels in competitive and noncompetitive immunoassay and in other aspects that are evident from the teachings presented herein or would be known to the skilled artisan are also contemplated.
A xe2x80x9cnatural polymerxe2x80x9d for the purpose of the present invention is herein defined as one that originates from a biological source including but not limited to: microorganisms, fungi, viruses, human, cow, pig, mouse, cat, dog, rat, or insect. Such natural polymers include proteins, peptides, glycoproteins, lipoproteins and recombinant and chemically modified species thereof, polysaccharides, celluloses, collagens, and latexes. Somewhat more specific examples include, dextrans, porcine, human, mouse, rat and bovine serum albumins or globulins, strepavidin, antibodies, enzymes such as peroxidase, xcex2-galactosidase, and alkaline phosphatase.
A xe2x80x9csynthetic polymerxe2x80x9d is defined herein as a polymer that does not necessarily directly originate from a biological source. It is one that is prepared by methods well known to the skilled artisan. For example, by way of monomer condensation using emulsion polymerization, ionic chain polymerization, carbonyl polymerization, radical chain polymerization and the like. It includes homopolymers such as polyacrylamides, polymethacrylates, polystyrenes, substitited polyacrylamides, polymethacrylates, and polystyrenes, and copolymers comprising two or more different monomeric units, such as acrylamide or substituted acrylamide, styrene and substituted styrene, and the like, as would be well known to one skilled in the art. It includes blockcopolymers, graftcopolymers, aqueous soluble and aqueous insoluble polymers and covalent and non-covalent combinations with natural polymers.
The term xe2x80x9clabelxe2x80x9d as defined herein includes: chemical elements, compounds, and enzymes that are capable of being detected directly or indirectly using, for example, absorption, fluorescence, or reflectance spectrophotometry, or radiation detection methods. A label may be a natural or synthetic polymer. For example, horseradish peroxidase is both a label and a natural polymer. But a label is not necessarily a natural or synthetic polymer.
A label capable of direct detection is one that is intrinsically capable of producing a detectable signal. Such labels include organic and inorganic substances capable of fluorscence, or phosphoresence, such as but not limited to fluorescein, and derivatives thereof, and N-(3-fluoranthyl)-maleimide, radionucleides, such as carbon 14, tritium and phosporus 32, and the like. Included are substances having appropriate spectral absorption such as but not limited to, azo-oxo, azo-tetrazo, azine, oxazine, thiazine, quinoline, indamine, pyrone and pyrazolone dyes.
A label that is capable of indirect detection requires the presence of one or more additional substances for production of the detectable signal. Such labels typically include but are not limited to enzymes that require the presence of a substrate(s), co-factor(s), metal(s) and the like. Peroxidases, most particularly, horseradish peroxidase, a common label, requires an electron donor and an oxidizing agent, such as luminol, di- or triarylimidazole leucodyes and hydrogen peroxide to produce a chemiluminescent product or dye, respectively. Other enzymes, such as xcex2-galactosidase, glucose oxidase and alkaline phosphatase, and the like, are also contemplated.
In general, labels include radioactive tags, enzymes, chromophores, fluorophores, stable free radicals, and enzyme cofactors, inhibitors and allosteric effectors.
A xe2x80x9creducing agentxe2x80x9d, for the purpose of the present invention, is any compound or reagent admixture that is capable of hydrogenating a double bond, such as carbon-carbon, carbon-nitrogen, carbon-oxygen and carbon-sulfur double bonds.
Useful reducing agents include but are not limited to: aluminum hydride, lithium aluminum hydride, borohydride and salts thereof. Catalytic hydrogenation over paladium, platinum or nickel or other hydrogenation methods can also be used. Sodium borohydride is a preferred reducing agent.
A xe2x80x9clinking groupxe2x80x9d is defined herein, as a chemical group comprising one or more atoms. A linking group connects one molecule to another, such as a natural polymer to a natural polymer, a synthetic polymer to a synthetic polymer, a natural polymer to a synthetic polymer, a label to a natural or synthetic polymer, a label to reduced cortisol, a label to cortisol, and so on, through formation of a covalent bond with each of the molecules it joins.
The linking group can comprise a substituted or unsubstituted straight chain or branched alkyl or heteroalkyl, such as oxyalkyl, thioalkyl, aminoalkyl, substituted or unsubstituted alkenyl, one or more substituted or unsubstituted hydrocarbon heterocyclic rings, one or more substituted or unsubstituted aryl or heteroaryl rings such as but not limited to, imidazoyl, isoxazolyl, pyridyl, piperidyl, piperazinyl, pyrazolyl,triazolyl, oxadiazolyl, pyridazinyl, pyrimidyl, pyrazinyl, quinolinyl, and quinazolinyl.
Linking or coupling of macromolecules, such as natural and synthetic polymers to other macromolecules, or linking small molecules, such as cortisol or analogs of cortisol, to macromolecules is well known in the art. Specifically with respect to cortisol, carbon 3(C3)is reactive with nucleophiles including, amines, oximes, and thio-oximes and others known in the art. The nucleophilic species may be a coupling (linking) agent, a label, a natural or synthetic polymer. If it is a coupling agent or other species having a reactive functional group (a label, etc.), upon covalently bonding to the C3 of cortisol it may be reacted further with another coupling agent or other species having an appropriately reactive functional group. Cortisol so derivatized can be used as the starting point for preparing the reduced cortisol compounds of the instant invention. Details of coupling chemistry and linking groups may be found in numerous publications, including, U.S. Pat. Nos. 3,654,090; 3,791,932; 3,875,011; 4,016,043; 4,040,907; 4,092,479; 4,213,894; 4,243,749; 4,376,165; 4,410,643; 4,752,658; 4,828,978; 4,879,249; 4,997,772; 5,053,497; 5,106,732; 5,147,777; 5,155,166; 5,162,219; 5,177,023; 5,284,948; 5,298,403; 5,308,749; 5,374,516; 5,391,483; 5,397,695; 5,401,633; 5,527,709; 5,543,311; 5,578,457; 5,652,346; 5,763,588; 5,770,390 and references identified therein; Yoshitake et al. Eur. J. Biochem., 101, 395, (1979) and Tjssen, in Laboratory Techniques in Biochemistry and Molecular Biology, pp 221-278 (1985) and references therein.
In brief, a linking group and molecule to which it is covalently attached can be connected through amide, ester, ether, thioester, and disulfide bonds. For example coupling or linking chemistry that includes reacting the molecules to be coupled with condensing agents such as carbodiimides, maleimides, ethylchloroformate, and glutaraldehyde is well known in the art.
The term xe2x80x9csamplexe2x80x9d refers to any substance that may contain the analyte of interest. A sample can be a biological fluid, such as cerebral spinal fluid, semen, vaginal secretions, sputum, ascites fluid, lacrimal fluid, sweat, serum, plasma, urine, whole blood or whole blood components including red and white blood cells, platelets, and other fluids or tissues of the body that may contain the analyte of interest. Optionally, samples may be obtained from water, soil and plants.
Immunoassays, which take advantage of natural immunological reactions, have found wide-spread use as analytical techniques in clinical chemistry. Because of the specificity of the reactions, they are particularly advantageous in quantifying biological analytes, that are present in very low concentration in biological fluids. Such analytes include, for example, antibodies, therapeutic drugs, narcotics, enzymes, hormones, proteins, etc.
In competitive binding immunoassays, a labeled analyte (the term includes immunocompetent analogs of the analyte) is placed in competition with unlabeled analyte for reaction with a fixed amount of an appropriate receptor, which is often immobilized on a solid susbstrate, or is capable of immobilization thereto. The labeled analyte that is bound to the receptor is separated from free labeled analyte. Unknown concentrations of the analyte can be determined from the measured signal of either the bound or free labeled analyte. The reaction proceeds as follows:
Analyte+labeled analyte+receptor⇄analytexe2x88x92receptor+labeled analytexe2x88x92receptor.
Immunoassays can be carried out in solution, in test devices where soluble and insoluble components can be separated, or in dry analytical elements. Immunoassays can be heterogeneous or homogeneous as those terms are known in the art. In heterogeneous assays, bound and free labeled immunoreactants (labeled analyte or labeled receptor for an analyte) are separated prior to signal measurement; whereas, in homogeneous assays separation of free from bound labeled immunoreactant is not required. The reduced cortisol conjugates of the instant invention can used in both homogeneous and heterogeneous assays.
Numerous publications relating to immunoassays and immunoassay methods, which include many of the above-cited publications relating to linking groups and coupling chemistry, are available to the practitioner. Additional publications include: U.S. Pat. Nos. 4,372,745; 4,670,381; 4,483,921; 4,517,288; 4,822,747; 4,824,778; 4,829,012; 4,839,299; 4,847,194; 4,847,195; 4,853,335; 4,855,226; 4,857,453; 4,857,454; 4,859,610; 4,863,876; 4,868,106; 4,868,130; 4,879,219; 5,663,054; 5,776,933 and all references cited therein; and Immunoassays in the Clinical Laboratory, Nakamura et al, eds., Alan R. Liss, Inc., (1979); Quantitative Enzyme Immunoassay, Engvall et al., eds, Blackwell Scientific Publications, (1978; Clinical Chemistry, Sommer et al., v.32,p. 1770-1774, (1986); Clinical Chemistry, Sommeret al., p 201-206 (1990); A Primer for Multilayer Immunoassay, Berke, American Chemical Society Conference Proceeding, p.303-312, Plenum Press, (1988); and all references cited therein.
In competitive immunoassays labeled analyte and sample containing free analyte can be added simultaneously or separately to an admixture comprising immobilized or immobilizable receptor that binds the analyte.
In the case of dry analytical elements, labeled analyte and immobilized receptor when present together in the element prior to contact with sample, are preferably present in separate zones.
Conventional materials and means for assembling dry-film analytical elements are described, for example, in U.S. Pat. Nos. 3,867,258; 3,992,158; 4,042,435; 4,050,898; 4,066,403; 4,153,668; 4,258,001; 4,292,272 and 4,430,436.
Methods to obtain antibodies that bind a specific molecule by immunizing suitable host animals is well known. Such methods are well documented and are described, for example, in the following publications: Methods in Immunology, Garvey,J. S., Cremer,N. E. and Sussdorf,D. H., W. A. Benjamin,Inc., Third Ed. (1977)and Handbook of Experimental Immunology, edited by Weir,D. M., Blackwell Scientific Publications, Third Ed., (1978)
Methods of producing hybridoma cell lines for secretion of antibodies is also well known, and are provided, for example, in U.S. Pat. Nos. 4,950,592; 5,338,671 and 5,650,324.
Preparation of HRP Labeled Reduced Cortisol-3-CMO-BSA
A method is provided below illustrating the preparation of a horseradish peroxidase (HRP) labeled reduced cortisol conjugate. In this method the reduction of a bovine serum albumin cortisol oxime conjugate (Cortisol-3-CMO-BSA) with sodium borohydride was carried out prior to conjugation with HRP; however, reduction of cortisol can be carried out subsequent to coupling with HRP.
There are three sites for reduction of Cortisol-3-CMO-BSA that are marked by an asterisk in the structure shown below 
Any or all of these sites can be reduced by treatment with a reducing agent or reducing admixture that is capable of hydrogenating a double bond, yielding products having a single site reduced as in the structures shown below. 
as well as, compounds having any two of the sites reduced, yielding three different reduced species, and all three sites reduced, as is readily apparent to the skilled artisan. Other conjugates comprising cortisol, which upon reduction produce the compounds generically defined in structures 1A, 1B, 1C and 1D, analogously will form different reduced cortisol species as exemplified above with Cortisol-3-CMO-BSA. Compounds having a covalent bond to C3 of cortisol that is not capable of being hydrogenated will be reduced only at the ring carbon-carbon double bond and/or the carbon-oxygen double bond (carbonyl group) of cortisol. All reduced forms individually or in any combination are contemplated in the present invention.